The phytoestrogen alpha-zearalenol reverses endothelial dysfunction induced by oophorectomy in rats

It has been shown recently that alpha-zearalenol, a resorcyclic acid lactone, prevents bone loss in a rat model of postmenopausal bone loss. We have therefore investigated the effects of this phytoestrogen on endothelial dysfunction induced by estrogen deficiency in rats. Female mature Sprague-Dawley rats underwent a bilateral oophorectomy (OVX rats). Sham-operated animals (sham OVX rats) were used as controls. Three weeks after surgery, animals were randomized to the following treatments: alpha-zearalenol (1 mg/kg/day, i.m., for 4 weeks), 17beta-estradiol (20 microg/kg/day, i.m., for 4 weeks), or their vehicle (100 microl, i.m., of cottonseed oil). Two other groups of rats were treated with alpha-zearalenol or 17beta-estradiol plus the pure estrogen receptor antagonist ICI 182780 (2.5 mg/kg/day, i.m., for 4 weeks). Mean arterial blood pressure (MAP), heart rate (HR), total plasma cholesterol, plasma estradiol, and plasma alpha-zearalenol were studied. We also investigated endothelial-dependent (acetylcholine, 10 nM to 10 microM) and endothelial-independent (sodium nitroprusside, 15 nM to 30 nM) relaxation of aortic rings, as well as N(G)-methyl-L-arginine (L-NMA: 10 to 100 microM)-induced vasoconstriction and calcium-dependent nitric oxide synthase (cNOS) activity in homogenates of lungs taken from both sham OVX rats and OVX rats. Untreated OVX rats had, compared with sham OVX animals, unchanged body weight, MAP, HR, and plasma cholesterol. In contrast oophorectomy reduced plasma estradiol levels (OVX, 2 +/- 0.5 pg/ml; sham OVX, 35 +/- 6 pg/ml), impaired endothelial-dependent relaxation and blunted L-NMA-induced contraction (L-NMA 100 microM: sham OVX, 2.7 +/- 0.3 g/mg tissue; OVX, 1.3 +/- 0.1 g/mg tissue). Moreover OVX rats showed a reduced calcium-dependent NO synthase (cNOS) activity. Treatment with alpha-zearalenol or with 17beta-estradiol reverted the endothelial dysfunction and increased cNOS activity in lung homogenates. These effects were abolished by the pure estrogen receptor antagonist ICI 182780. Our data suggest that alpha-zearalenol improves endothelial-dependent relaxation in OVX rats through an estrogen receptor-mediated effect.     

A novel Ca(2+) influx pathway in mammalian primary sensory neurons is activated by caffeine.

Single-cell microfluorimetry and electrophysiology techniques were used to identify and characterize a novel Ca(2+) influx pathway in adult rabbit vagal sensory neurons. Acutely dissociated nodose ganglion neurons (NGNs) exhibit robust Ca(2+)-induced Ca(2+) release (CICR) that can be triggered by 10 mM caffeine, the classic agonist of CICR. A caffeine-induced increase in cytosolic-free Ca(2+) concentration ([Ca(2+)](i)) is considered diagnostic evidence of the existence of CICR. However, when CICR was disabled through depletion of intracellular Ca(2+) stores or pharmacological blockade of intracellular Ca(2+) release channels (ryanodine receptors), caffeine still elicited a significant rise in [Ca(2+)](i) in approximately 50% of NGNs. The same response was not elicited by pharmacological agents that elevate cyclic nucleotide concentrations. Moreover, extracellular Ca(2+) was obligatory for such caffeine-induced [Ca(2+)](i) rises in this population of NGNs, suggesting that Ca(2+) influx is responsible for this rise. Simultaneous microfluorimetry with whole cell patch-clamp studies showed that caffeine activates an inward current that temporally parallels the rise in [Ca(2+)](i). The inward current had a reversal potential of +8.1 +/- 6.1 (SE) mV (n = 4), a mean peak amplitude of -126 +/- 24 pA (n = 4) at E(m) = -50 mV, and a slope conductance of 1.43 +/- 0.79 nS (n = 4). Estimated EC(50) values for caffeine-induced CICR and for caffeine-activated current were 1.5 and approximately 0.6 mM, respectively. These results indicate that caffeine-induced rises in [Ca(2+)](i), in the presence of extracellular Ca(2+), can no longer be interpreted as unequivocal diagnostic evidence for CICR in neurons. These results also indicate that sensory neurons possess a novel Ca(2+) influx pathway.     

Vagotomy decreases excitability in primary vagal afferent somata

Standard patch-clamp and intracellular recording techniques were used to monitor membrane excitability changes in adult inferior vagal ganglion neurons (nodose ganglion neurons, NGNs) 5 days following section of the vagus nerve (vagotomy). NGNs were maintained in vivo for 5 days following vagotomy, and then in vitro for 2-9 h prior to recording. Vagotomy increased action potential (AP) threshold by over 200% (264 +/- 19 pA, mean +/- SE, n = 66) compared with control values (81 +/- 20 pA, n = 68; P < 0.001). The number of APs evoked by a 3 times threshold 750-ms depolarizing current decreased by >70% (from 8.3 to 2.3 APs, P < 0.001) and the number of APs evoked by a standardized series of (0.1-0.9 nA, 750 ms) depolarizing current steps decreased by over 80% (from 16.9 APs to 2.6 APs, P < 0.001) in vagotomized NGNs. Similar decreases in excitability were observed in vagotomized NGNs in intact ganglia in vitro studied with "sharp" microelectrode techniques. Baseline electrophysiological properties and changes following vagotomy were similar in right and left NGNs. A "sham" vagotomy procedure had no effect on NGN properties at 5 days, indicating that changes were due to severing the vagus nerve itself, not surrounding tissue damage. NGNs isolated after being maintained 17 h in vivo following vagotomy revealed no differences in excitability, suggesting that vagotomy-induced changes occur some time from 1-5 days after injury. Decreased excitability was still observed in NGNs isolated after 20-21 days in vivo following vagotomy. These data indicate that, in contrast to many primary sensory neurons that are thought to become hyperexcitable following section of their axons, NGNs undergo a marked decrease in electrical excitability following vagotomy.     

Chemical communication between vagal afferent somata in nodose Ganglia of the rat and the Guinea pig in vitro

The cell bodies of spinal afferents, dorsal root ganglion neurons, are depolarized several millivolts, and their probability of spiking increased when axons of neighboring somata in the same ganglion are electrically stimulated repetitively. This form of neural communication has been designated cross-depolarization (CD) and cross-excitation (CE). The existence of CD and CE between somata of vagal afferents (nodose ganglion neurons, NGNs) of rats and guinea pigs was investigated by electrically stimulating the vagus nerve while recording the electrical activity of NGNs in intact nodose ganglia with sharp intracellular microelectrodes. CD and CE in NGNs were manifested by a membrane depolarization (approximately 4 mV), the presence of spontaneous action potentials, and a decreased spike threshold. CD was dependent on the frequency and intensity of vagal nerve stimulation. Two distinct types of CD were observed: 1) in NGNs with large input resistances (R(in)), CD was dependent on [Ca2+]o, associated with increased membrane conductance, and had an extrapolated reversal potential (E(rev)) value of about -25 mV; and 2) in NGNs with low R(in), CD was independent of [Ca2+]o, not accompanied by a membrane conductance change, or a measurable E(rev) value. These data reveal the existence of a chemical communication pathway between vagal afferent somata and suggest the possibility that communication between different visceral organs may occur at the level of the primary vagal afferent neuron.     

Chronic estrogen deficiency leads to molecular aberrations related to neurodegenerative changes in follitropin receptor knockout female mice

The follitropin receptor knockout (FORKO) mouse undergoes ovarian failure, thereby providing an animal model to investigate the consequences of the depletion of circulating estrogen in females. The estrogen deficiency causes marked defects in the female reproductive system, obesity, and skeletal abnormalities. In light of estrogen's known pleiotropic effects in the nervous system, our study examined the effects of genetically induced estrogen-testosterone imbalance on this system in female FORKO mice. Circulating concentrations of 17-beta-estradiol (E2) in FORKO mice are significantly decreased (FORKO -/-: 1.13+/-0.34 pg/ml; wild-type +/+: 17.6+/-3.5 pg/ml, P<0.0001, n=32-41); in contrast, testosterone levels are increased (-/-: 37.7+/-2.3 pg/ml; wild-type +/+: 3.9+/-1.7 pg/ml, P<0.005, n=25-33). The focus was on the activities of key enzymes in the central cholinergic and peripheral nervous systems, on dorsal root ganglia (DRGs) capacity for neurite outgrowth, and on the phosphorylation state of structural neurofilament (NF) proteins. Choline acetyltransferase activity was decreased in several central cholinergic structures (striatum 50+/-3%, hippocampus 24+/-2%, cortex 12+/-3%) and in DRGs (11+/-6%). Moreover, we observed aberrations in the enzymatic activities of mitogen-activated protein kinases (extracellular-regulated kinase and c-Jun N-terminal kinase) in the hippocampus, DRGs, and sciatic nerves. Hippocampal and sensory ganglia samples from FORKO mice contained hyper-phosphorylated NFs. Finally, explanted ganglia of FORKO mice displayed decreased neurite outgrowth (20-50%) under non-treated conditions and when treated with E2 (10 nM). Our results demonstrate that genetic depletion of circulating estrogen leads to biochemical and morphological changes in central and peripheral neurons, and underlie the importance of estrogen in the normal development and functioning of the nervous system. In particular, the findings suggest that an early and persisting absence of the steroid leads to neurodegenerative changes and identify several key enzymes that may contribute to the process. This model provides a system to explore the consequences of circulating estrogen deprivation and other hormonal imbalances in the nervous system.     

Localized IP3-evoked Ca2+ release activates a K+ current in primary vagal sensory neurons

Electrophysiological and microfluorimetric techniques were used to determine whether intracellular photorelease of caged IP(3), and the consequent release of Ca(2+), could trigger a Ca(2+)-activated K(+) current (I(IP3)). Photorelease of caged IP(3) evoked an I(IP3) that averaged 2.36 +/- 0.35 (SE) pA/pF in 24 of 28 rabbit primary vagal sensory neurons (nodose ganglion neurons, NGNs) voltage-clamped at -50 mV. I(IP3) was abolished by intracellular BAPTA (2 mM), a Ca(2+) chelator. Changing the K(+) equilibrium potential by increasing extracellular K(+) ion concentration caused a predicted Nernstian shift in the reversal potential of I(IP3). These results indicated that I(IP3) was a Ca(2+)-dependent K(+) current. I(IP3) was unaffected by three common antagonists of Ca(2+)-activated K(+) currents: bath-applied iberiotoxin (50 nM) or apamin (100 nM), and intracellular 8-Br-cAMP (100 microM) included in the patch pipette. We have previously demonstrated that both IP(3)-evoked Ca(2+) release and Ca(2+)-induced Ca(2+) release (CICR) are co-expressed in NGNs and that CICR can trigger a Ca(2+)-activated K(+) current. In the present study, using caffeine, a CICR agonist, to selectively attenuate intracellular Ca(2+) stores, we showed that IP(3)-evoked Ca(2+) release occurs independently of CICR, but interestingly, that a component of I(IP3) requires CICR. These data suggest that IP(3)-evoked Ca(2+) release activates a K(+) current that is pharmacologically distinct from other Ca(2+)-activated K(+) currents in NGNs. We describe several models that explain our results based on Ca(2+) signaling microdomains in NGNs.     

Neurochemical phenotype of vagal afferent neurons activated to express C-FOS in response to luminal stimulation in the rat

The vagus nerve conveys meal-induced primary afferent responses to the brainstem. Electrophysiological studies indicate that luminal stimuli such as osmolarity and the digestion products of carbohydrates elicit powerful vagal nodose neuronal responses by activating serotonin 3 (5-hydroxytryptamine-3, 5-HT3) receptors on intestinal mucosal afferent fibers. To characterize the neurochemical phenotype of neurotransmitters in vagal nodose neurons that are activated by luminal stimulation, we examined c-fos protein (c-Fos) expression in response to luminal stimulation in conscious rats. A double-labeling technique using antisera to glutamate (Glu), substance P (SP), calcitonin gene-related peptide (CGRP), and somatostatin (SS) was used to determine the neurochemical profile of c-Fos-positive neurons. c-Fos immunoreactivity was insignificant in vehicle-treated rats. Luminal perfusions of NaCl (500 mOsm), tap water (5 mOsm), maltose (300 mmol/l), and 5-HT (10(-5) mol/l) each elicited a significant increase in the number of cells expressing c-Fos. Chronic vagotomy eliminated an increase in nodose neuronal c-Fos expression, and the 5-HT3 receptor antagonist granisetron significantly reduced it. Glu-, SP-, and CGRP-containing neurons represented 28%, 53%, and 19%, respectively, of the total population of nodose neurons. Few neurons contained SS. Double-labeling studies revealed that of the c-Fos-positive neurons responsive to hypertonic NaCl, 52%, 41%, and 3% exhibited immunoreactivity for Glu, SP, and CGRP, respectively. Of those responsive to tap water, 47%, 50%, and 4% exhibited immunoreactivity for Glu-, SP- and CGRP, respectively. In addition, 44%, 38%, and 8% of 5-HT-stimulated and 30%, 32%, and 5% of maltose-stimulated c-Fos-positive neurons exhibited, respectively, Glu, SP, and CGRP immunoreactivity. The few neurons that contained SS did not express c-Fos. CONCLUSIONS: Vagal primary afferent neurons that respond to 5-HT-dependent luminal stimuli, such as hyperosmolarity and maltose, contain mainly Glu and SP. These neurons appear to play an important role in the mediation of the vago-vagal reflex elicited by luminal stimuli.     

Cytokines in symptomatic asthma airways.

To determine whether cytokines are generated in vivo in subjects with asthma, we have measured cytokine levels (tumor necrosis factor [TNF], granulocyte-macrophage-colony-stimulating factor [GM-CSF], interleukin [IL]-1 alpha, IL-1 beta, IL-2, IL-4, and IL-6) in the airways of subjects with symptomatic (N = 24) and asymptomatic (N = 9) asthma with immunoassays (GM-CSF, IL-1 alpha, IL-1 beta, IL-2, and IL-4) or bioassays (TNF and IL-6) and the polymerase chain reaction (IL-1 beta and TNF). Significant levels of TNF (578 +/- 917 pg/ml versus 24 +/- 29 pg/ml) (p = 0.01), GM-CSF (24 +/- 41 pg/ml versus less than 8 pg/ml) (p = 0.02), and IL-6 (225 +/- 327 pg/ml versus 7 +/- 12 pg/ml) (p = 0.01), but not IL-1 alpha or IL-4, were detected in the bronchoalveolar lavage fluid (BALF) of patients with symptomatic compared with BALF of patients with asymptomatic asthma. Levels of IL-1 beta (266 +/- 270 pg/ml versus less than 20 pg/ml) (p = 0.001) and IL-2 (1.4 +/- 2.8 ng/ml versus less than 0.3 ng/ml) (p = 0.05) in BALF in patients with symptomatic compared with that in BALF levels in patients with asymptomatic asthma suggested activation of alveolar macrophages and T cells. Thus, in episodes of asthma, several cytokines, including TNF, GM-CSF, IL-1 beta, IL-2, and IL-6 are detectable in BALF.     

Chronic smoking enhances tachykinin synthesis and airway responsiveness in guinea pigs

This study tests the hypothesis that the bronchial hyperreactivity induced by chronic cigarette smoke (CS) exposure involves the increased expression and release of tachykinins and calcitonin gene-related peptide (CGRP) from afferent nerve fibers innervating the airways. In guinea pigs chronically exposed to CS (20 min twice daily for 14-17 d), peak response in total lung resistance to capsaicin (1.68 microg/kg, intravenously) was significantly greater than that evoked by the same dose of capsaicin in control (air-exposed) animals. This augmented response in CS-exposed animals was abolished after treatment with CP-99994 and SR-48968, the neurokinin (NK)-1 and NK-2 receptor antagonists, suggesting the involvement of tachykinins in chronic CS-induced airway hyperresponsiveness (AHR). Further, substance P (SP)-like immunoreactivity (LI) and CGRP-LI in the airway tissue were significantly greater in the CS animals than in the control animals. Finally, beta-preprotachykinin (PPT, a splice variant from the PPT A gene encoding tachykinins including SP and NKA) messenger RNA levels as measured by in situ hybridization histochemistry displayed a significant increase in jugular ganglion neurons but not in dorsal root or nodose ganglion neurons. These data suggest that chronic CS-induced AHR is related to an increase in SP synthesis and release in jugular ganglion neurons innervating the lungs and airways.     

No difference in serum leptin concentrations between urban-dwelling Austronesians and Non-Austronesians in Papua New Guinea.

Pacific Islands populations can be broadly divided into Austronesians (AN) and Non-Austronesians (NAN); obesity and type 2 diabetes are prevalent in the former, although leptin levels in both groups have seldom been investigated. Thirty-seven (20 male and 17 female) adult pairs, matched by age and percent body fat, from AN-speaking Balopa and NAN-speaking Huli, all of whom migrated to settle in Port Moresby, the capital of Papua New Guinea, were selected for comparison of their serum leptin concentrations. The Balopa did not differ significantly from the Huli in age (30.5 +/- 9.7 and 30.0 +/- 8.7 years for males, 33.7 +/- 8.9 and 34.1 +/- 7.5 years for females, respectively) or percent body fat (19.4 +/- 5.6 and 18.8 +/- 4.6 for males, 34.1 +/- 6.2 and 33.3 +/- 5.0 for females), although the BMI of females was lower in the Balopa (26.4 +/- 4.9) than in the Huli (29.7 +/- 4.7) (P = 0.02). In both ethnic groups, females had markedly higher leptin concentrations than males, but there was no significant inter-group difference in males (3.5 +/- 2.6 and 3.1 +/- 4.7 ng/ml, P = 0.14) or females (22.7 +/- 12.9 and 19.7 +/- 11.9 ng/ml, P = 0.40), after controlling for lifestyle factors and serum lipids. Multiple regression analysis revealed that significant predictors of leptin concentration were % body fat (beta = 0.58), sex (male, 0; female, 1; beta = 0.27), and smoker status (non-smoker, 0; smoker, 1; beta = -0.15) (R(2) = 0.80), implying that the leptin concentration was primarily determined by lifestyle-derived body fatness. In conclusion, the NAN populations do not endogenously differ in leptin status from the AN populations, who have been recognized as a typical group with a "thrifty" genotype.     

Determination of tumor necrosis factor-alpha levels in dengue virus infected patients by sensitive biotin-streptavidin enzyme-linked immunosorbent assay

A modified sandwich enzyme-linked immunosorbent assay using biotin-streptavidin system (BS-ELISA) was developed to determine levels of tumor necrosis factor-alpha (TNF-alpha) in serum samples of children infected with dengue virus (n=99) and healthy controls (n=41). The minimum detectable concentration of TNF-alpha by the BS-ELISA was 3.3 pg/ml. The mean TNF-alpha level was highest in those patients with dengue shock syndrome (DSS) or dengue hemorrhagic fever (DHF) grade III (37.44+/-42.0 pg/ml). Lower levels were found in DHF grade I (28.44+/-42.7 pg/ml), DHF grade II (24. 21+/-25.4 pg/ml) and dengue fever (DF) (14.10+/-24.0 pg/ml). TNF-alpha in the sera of DF and DHF patients could be detected on days 2-6 after the onset of fever, the high level occurring on day 5. TNF-alpha was detected in 41.4% (24.01+/-35.2 pg/ml) of dengue virus infected patients and 7.3% (4.2+/-15.6 pg/ml) of control subjects. The sera of patients contained significantly higher levels of TNF-alpha than the sera of controls, P-value<0.001. DHF patients had significantly higher levels of TNF-alpha than DF patients (P-value=0.020) but no difference in the TNF-alpha levels from sera of DHF grades I-III patients was observed (P-value=0.295). The results indicate that the BS-ELISA is a very sensitive method for determining TNF-alpha in serum samples of DF and DHF patients. The TNF-alpha levels might be associated with dengue virus infection and related to disease severity of DHF.     

Rhythm of melatonin excretion in obstructive sleep apnea syndrome

Melatonin is a pineal hormone that regulates sleep and wake status. Melatonin concentrations in blood serum were measured using radioisotope method in 33 males (age 48 +/- 10) with obstructive sleep apnea syndrome. The following melatonin concentrations were measured: 54 +/- 72 pg/ml (9 p.m.) 424 +/- 838 pg/ml (2 a.m.) and 307 +/- 534 pg/ml (6 a.m.). In patients with high peak melatonin concentration (> 200 pg/ml) as compared with the patients with low peak melatonin concentration (< 200 pg/ml) there were higher index of respiratory disorders during sleep (53 +/- 18 vs 38 +/- 20, p < 0.05) and lower minimal SaO2 during sleep apnea (52 +/- 17% vs 70 +/- 10%, p < 0.05); they were also more tired in the morning and were more sleepy during the day (Epworth sleepiness scale 17 +/- 6 vs 11 +/- 6, p < 0.01). In 66% of patients peak melatonin concentration was observed at 2 a.m. In 24% of patients peak melatonin secretion was prolonged to early morning hours. CONCLUSIONS: In most of patients there is peak melatonin excretion at 2 a.m. Patients with high apnea and hypopnea index and daytime sleepiness have high peak melatonin concentrations.     

A comparison of tau protein in cerebrospinal fluid between corticobasal degeneration and progressive supranuclear palsy

Many clinical and pathological discussions have been focused on the difficulty of differential diagnosis between corticobasal degeneration (CBD) and progressive supranuclear palsy (PSP) in recent years. This study was conducted to evaluate the usefulness of tau proteins in cerebrospinal fluid (CSF) for the differentiation of these two diseases. Subjects consisted of 10 patients with CBD (four males and six females with a mean age of 67.9+/-5.8 years), 12 patients with PSP (eight males and four females with a mean age of 62.6+/-5.8 years) and 36 control subjects (CTL) (16 males and 20 females with a mean age of 65.8+/-9.9 years). The CBD group included patients with probable CBD, while all the patients in the PSP group satisfied the diagnostic criteria developed by the National Institute of Neurological Disorders and Stroke and Society for PSP (NINDS-SPSP). CSF tau proteins were measured with the sandwich ELISA method (Innogenetics, Belgium). The CSF tau protein level was 320.1+/-86.5 pg/ml in the CBD group, 151.5+/-52.7 pg/ml in the PSP group and 128.7+/-91.7 pg/ml in the CTL group. Significant differences were noted in tau protein levels between the CBD group and both the PSP group (P<0.001) and the CTL group (P<0.005). We suggested that the measurement of CSF tau proteins may be useful for the differentiation between CBD and PSP.     

Stress induces substance P in vagal sensory neurons innervating the mouse airways

BACKGROUND: Tachykinins-like substance P (SP) have been shown to play an important role in initiating and perpetuating airway inflammation. Furthermore, they are supposed to be released into tissues in response to stress. OBJECTIVE: The aim of this study was to investigate the effects of stress alone or in combination with allergic airway inflammation on SP expression in sensory neurons innervating the mouse airways. METHODS: Balb/c mice were systemically sensitized to ovalbumin (OVA), followed by allergen aerosol exposure, and compared with non-sensitized controls. Additionally, OVA-sensitized and -challenged and non-sensitized mice were exposed to sound stress. SP expression in airway-specific and overall vagal sensory neurons of the jugular and nodose ganglion complex was analysed using retrograde neuronal tracing in combination with immunohistochemistry. Preprotachykinin A (PPT-A) mRNA, the precursor for SP, was quantified in lung tissue by real-time PCR. Bronchoalveolar lavage (BAL) fluid was obtained, and cell numbers and differentiation were determined. RESULTS: Stress and/or allergic airway inflammation significantly increased SP expression in retrograde-labelled vagal sensory neurons from the mouse lower airways compared with controls [stress: 15.7+/-0.8% (% of retrograde-labelled neurons, mean+/-SEM); allergen: 17.9+/-0.4%; allergen/stress: 13.1+/-0.7% vs. controls: 6.3+/-0.3%]. Similarly, SP expression increased in overall vagal sensory neurons identified by the neuronal marker protein gene product (PGP) 9.5 [stress: 9.3+/-0.6% (% of PGP 9.5-positive neurons, means+/-SEM); allergen: 12.5+/-0.4%; allergen/stress: 10.2+/-0.4% vs. controls: 5.1+/-0.3%]. Furthermore, stress significantly increased PPT-A mRNA expression in lung tissue from OVA-sensitized and -challenged animals, and immune cells were identified as an additional source of SP in the lung by immunohistochemistry. Associated with enhanced neuronal SP expression, a significantly higher number of leucocytes were found in the BAL following allergen exposure. Further, stress significantly increased allergen-induced airway inflammation identified by increased leucocyte numbers in BAL fluids. CONCLUSION: The central event of sound stress leads to the stimulation of SP expression in airway-specific neurons. However, in sensitized stressed mice an additional local source of SP (probably inflammatory cells) might enhance allergic airway inflammation.     

17Beta-estradiol delivered by three different matrix patches 50 microg/day: a three way cross-over study in 21 postmenopausal women

The aim of this study was to investigate the systemic bioavailability and plasma profiles of 17beta-estradiol (E2) after the application of three matrix patches for the transdermal delivery of E2: Menorest, Tradelia, and Estraderm MX claiming to deliver a dosage of 50 microg E2/day. All three patches were each worn randomly by 21 postmenopausal women volunteers over a 4-day period (i.e. 96 h). Each of the three treatment periods were separated by an at least 7 day wash out period according to a randomized, 3-way crossover design. Blood samples were taken from the antecubital vein before and 3, 6, 9, 12, 24, 28, 33, 48, 57, 72, 81, and 96 h after application. E2 plasma values were determined by a specific direct radioimmunoassay method. The following pharmacokinetic parameters were evaluated: AUC0-96h, Cmax, Tmax, Cmin, C(average). The time to reach the maximal E2 value of 32 h was the only pharmacokinetic parameter which was identical for all three patches. Menorest produced the highest E2 bioavailability judged by the AUC0-96h = 3967.8 +/- 1651.8 pg/ml, C(average) = 41.3 +/- 21.3 pg/ml, Cmin = 36.8 +/- 8.6 pg/ml. Tradelia showed statistically not significantly smaller C(average) = 38.9 +/- 17.0 pg/ml, AUC0-96h = 3737.9 +/- 1637.6 pg/ml x per h, and Cmin = 33.8 +/- 26.7 than Menorest. Estraderm MX showed lowest E2 plasma profiles Cmax = 38.9 +/- 25.1 pg/ml, C(average) = 33.2 +/- 17.1 pg/ml, AUC0-96 = 3192.1 +/- 1646.0 pg/ml per x h. Menorest showed the smallest fluctuation over the entire test period, similar to Estraderm MX, while Tradelia showed the highest E2-fluctuation (P < 0.01): Tradelia exhibited the highest Cmax = 48.0 +/- 20.3 pg/ml. When E2 baseline levels, prior to patch application are subtracted individually from the produced E2 plasma level, Estraderm MX is not bioequivalent to Menorest (P < 0.05). A circadian curve pattern of the E2 plasma level was observed for all patches: in the evening higher E2 plasma level were always detected compared with the morning, however, less pronounced with Estraderm MX. Individual comparison of AUC0-96h of each patch exhibited a large interindividual variability of 2000-8000 pg/ml per h for all three patches but relatively small individual variability: women with high E2 bioavailability (high responders) maintained high bioavailability in all applied patches, women identified as low and medium responders remained the same regardless of the applied patch. Menorest produced in 2/3 of all postmenopausal women with the highest E2 bioavailability (AUC0-96h), Tradelia was found in less than 1/3 (28.6%), and Estraderm MX in only one postmenopausal woman. Menorest only produced the highest reduction in postmenopausal symptoms together with Tradelia. Estraderm MX produced a smaller reduction in postmenopausal symptoms compared to Menorest and Tradelia. The observed side-effects were approximately equal in all three patches, with a maximum value after 72 h. It can be concluded that the three patches for the transdermal delivery of E2 claiming to deliver 50 microg E2/day differed from each other in their pharmacokinetic performance, although statistically not significant: Menorest exhibited the highest C(average), AUC and Cmin, and the lowest fluctuation, followed by Tradelia and Estraderm MX.     

Thymosin alpha 1 and thymosin beta 4 in serum: comparison of normal, cord, homosexual and AIDS serum

Thymosin alpha 1 and thymosin beta 4 were first isolated from thymosin fr. 5 and have demonstrated biological activities on the immune system. They are chemically distinct and differ in their immunological activity profiles. The levels of thymosin alpha 1 and thymosin beta 4 were assessed by radioimmunoassay in the same serum samples. Normal thymosin alpha 1 levels were 670 +/- 163 pg/ml for males and 652 +/- 162 pg/ml for females. Normal thymosin beta 4 levels were 974 +/- 400 ng/ml for males and 889 +/- 345 ng/ml for females. No correlation between the levels of the peptides in serum from normal donors was observed. Although many samples of serum from neonates (cord blood), homosexuals and AIDS patients had elevated levels of one or both peptides, no correlation between the two peptides was found. Of potential significance is the observation that while thymosin alpha 1 and beta 4 are elevated in many individuals with AIDS (57 and 48% respectively), the individuals with AIDS related immune dysfunctions had predominantly elevated thymosin alpha 1 (54 vs 15%). These studies suggest that serum levels of the two peptides are modulated separately and that both are of potential value in defining the risk of individuals for developing AIDS.     

Levels of nonphosphorylated and phosphorylated tau in cerebrospinal fluid of Alzheimer's disease patients : an ultrasensitive bienzyme-substrate-recycle enzyme-linked immunosorbent assay

We have developed an ultrasensitive bienzyme-substrate-recycle enzyme-linked immunosorbent assay for the measurement of Alzheimer's disease (AD) abnormally hyperphosphorylated tau in cerebrospinal fluid (CSF). The assay, which recognizes attomolar amounts of tau, is approximately 400 and approximately 1300 times more sensitive than conventional enzyme-linked immunosorbent assay in determining the hyperphosphorylated tau and total tau, respectively. With this method, we measured both total tau and tau phosphorylated at Ser-396/Ser-404 in lumbar CSFs from AD and control patients. We found that the total tau was 215 +/- 77 pg/ml in cognitively normal control (n = 56), 234 +/- 92 pg/ml in non-AD neurological (n = 37), 304 +/- 126 pg/ml in vascular dementia (n = 46), and 486 +/- 168 pg/ml (n = 52) in AD patients, respectively. However, a remarkably elevated level in phosphorylated tau was only found in AD (187 +/- 84 pg/ml), as compared with normal controls (54 +/- 33 pg/ml), non-AD (63 +/- 34 pg/ml), and vascular dementia (72 +/- 33 pg/ml) groups. If we used the ratio of hyperphosphorylated tau to total tau of > or =0.33 as cutoff for AD diagnosis, we could confirm the diagnosis in 96% of the clinically diagnosed patients with a specificity of 95%, 86%, 100%, and 94% against nonneurological, non-AD neurological, vascular dementia, and all of the three control groups combined, respectively. It is suggested that the CSF level of tau phosphorylated at Ser-396/Ser-404 is a promising diagnostic marker of AD.     

Prevention of age-related dysregulation of calcium dynamics by estrogen in neurons

To determine the impact of aging and 17beta-estradiol on neuronal Ca2+ homeostasis, intracellular Fura-2 Ca2+-imaging was conducted during 20-pulses of glutamate in hippocampal neurons cultured from embryonic (E18), middle-age (10 months) and old (24 months) rat brain. Marked age-related differences in intracellular Ca2+ ([Ca2+]i) homeostasis and striking regulation by 17beta-estradiol were seen. Embryonic neurons exhibited the greatest capacity to regulate Ca2+ homeostasis followed by middle-age neurons. In old neurons, the first peak [Ca2+]i was substantially greater than at other ages and the return to baseline Ca2+ rapidly dysregulated with an inability to restore [Ca2+]i following the first glutamate pulse which persisted throughout the 20 pulses. 17beta-Estradiol pretreatment of old neurons profoundly attenuated the peak [Ca2+]i rise and delayed the age-associated dysregulation of baseline [Ca2+]i, normalizing responses to those of middle-age neurons treated with estradiol. The efficacy of 17beta-estradiol extended below 10 pg/ml with full protection against toxicity from glutamate and Abeta (1-40). These results demonstrate age-associated dysregulation of [Ca2+]i homeostasis which was largely prevented by 17beta-estradiol with implications for estrogen/hormone therapy.     

Immune-regulating Th1- and Th2-cytokines in chronic infections caused by hepatitis B and C viruses

The serum levels of Th1 (gamma-IFN and sIL-2r) and of Th2 (IL-10) cytokines were measured in 33 patients (23 males and 10 females, mean age 23.1 +/- 1.9) with chronic viral hepatitis (CVH) according to a disease etiology (6 patients with hepatitis B--CVHB, 15 patients with hepatitis C--CVHC, and 12 patients with a mixed form of chronic hepatitis B and C--HBV + HCV). Besides, the contents of the studied cytokines were compared with the traditional infection markers and the presence of viremia. The similar indices taken from 10 healthy persons served as controls. The concentration of gamma-IFN was found to be reliably higher (p < 0.05) in patients of all three groups (0.32 +/- 0.07, 0.34 +/- 0.09 and 0.25 +/- 0.06 pg/ml, respectively) regardless of a disease etiology and as compared with the control value (0.09 +/- 0.04) pg/ml). At the same time, the levels of gamma-IFN, sIL-2r and IL-10 (0.25 +/- 0.06 pg/ml, 166.5 +/- 31.3 IU/ml and 48.1 +/- 8.4 pg/ml, respectively) was found to be reliably (p < 0.05 and p < 0.01) higher, as compared to the controls (0.09 +/- 0.04, 57.1 +/- 5.6 and 10.8 +/- 7.8, respectively), only in patients with the mixed infection of hepatitis. Like in our previous study, a trend was established towards the growing mean values of the IL-r level from its lowest parameters in the group of CVHB patients towards its highest parameters in the group with the mixed hepatitis form. According to our data, the IL-2r level correlated reliably with the activity of AlAt (r = 0.452; p < 0.05), while the gamma-IFN content correlated reliably with the IL-10 concentration (r = 0.805; p < 0.05), and the gamma-IFN content correlated with the IL-10 concentration (r = 0.805; p < 0.01) irrespective of disease pathology.     

The effect of trimetazidine on C-reactive protein, cytokines and adhesion molecules in the course of acute myocardial infarction

The aim of this randomised, double-blind, placebo controlled, parallel group study was to assess the effect of trimetazidine (TMZ), a potent antiischaemic drug, on plasma C-reactive protein (C-RP), cytokine and adhesion molecule levels. The study population consists of 18 patients (16 males, 2 females, average age 56.45 +/- 10.97 years) with acute myocardial infarction admitted within 6 hours after onset of symptoms and treated with streptokinase. Blood samples were taken at 3-hour intervals during the time of treatment. All patients were randomised blindly using a centralised randomisation process, between trimetazidine (40 mg bolus i.v. then 60 mg per day for 48 hours intravenously in glucose infusion) or placebo group. Plasma C-RP level was significantly lower in TMZ group (39.5 mg/ml +/- 9.7 mg/ml) as compared to placebo (75.7 +/- 29.4 mg/ml, p < or = 0.001) and peaked 28 hours later in TMZ group. Plasma interleukin 6 (IL 6) level showed a sharp peak 9 hours after the onset of the symptoms in TMZ group (116.9 +/- 180.2 pg/ml vs. 45.4 +/- 37.9 pg/ml) and was increased up to 30 hours after the onset of the symptoms. Plasma interleukin 1 beta (IL 1 beta) was also higher in TMZ group notably 21 hours after the onset of symptoms (26.4 +/- 9.3 pg/ml vs. 16.2 +/- 2.4 pg/ml). TMZ group showed lower plasma E-selectin levels. Plasma IL 8, TNF alpha and ICAM 1 levels were without statistical significant differences. The present study demonstrates a significant reduction of plasma C-reactive protein level in the course of acute myocardial infarction treated with streptokinase and intravenous trimetazidine infusion compared with the group of patients without trimetazidine treatment.